Catalytic properties of 2,3-dihydroxybiphenyl 1,2-dioxygenase from Dyella Ginsengisoli LA-4 immobilized on mesoporous silica SBA-15

Article abstract of DOI:10.1016/j.molcatb.2013.11.003

In this study, 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC) from Dyella ginsengisoli LA-4 was immobilized on the mesoporous silica SBA-15 in order to improve its stability with relatively high retaining activities. By Fourier transformed infrared spectros

Full text of DOI:10.1016/j.molcatb.2013.11.003

Journal of Molecular Catalysis B: Enzymatic 99 (2014) 136–142
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Catalytic properties of 2,3-dihydroxybiphenyl 1,2-dioxygenase from
Dyella Ginsengisoli LA-4 immobilized on mesoporous silica SBA-15
Yuanyuan Qu∗, Chunlei Kong, Hao Zhou, E Shen, Jingwei Wang, Wenli Shen,
Xuwang Zhang, Zhaojing Zhang, Qiao Ma, Jiti Zhou
State Key Laboratory of Fine Chemical and Industrial Ecology and Environmental Engineering (Ministry of Education, China), School of Environmental
Science and Technology, Dalian University of Technology, Dalian 116024, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC) from Dyella ginsengisoli LA-4 was immo-
bilized on the mesoporous silica SBA-15 in order to improve its stability with relatively high retaining
activities. By Fourier transformed infrared spectroscopy (FTIR) and N2 adsorption/desorption isotherms,
BphC was confirmed to be successfully adsorbed and captured on SBA-15. Under the experimental con-
ditions, the maximum loading amount could reach 124.6 mg protein g−1 support. The immobilized BphC
could keep 16% of its initial activity after being stored at 4 ◦C for 5 days, whereas the free enzyme lost
its activity rapidly within 12 h. Furthermore, the immobilized BphC possessed 90% of its initial activity
after incubating at 40 ◦C for 4 h, indicating that the thermostability of BphC was significantly improved
by immobilization. The biodegradation of catecholics compounds has been studied by immobilized BphC
in batch system. The enhanced properties of immobilized BphC were further analyzed by the circular
dichroism (CD), which supposed that the activity and stability alteration was due to the changes of sec-
ondary structure of BphC. This study would contribute to realizing the more effective biodegradation and
bioremediation processes by immobilizing enzymes on the mesoporous silica materials.
Received 23 September 2013
Received in revised form 4 November 2013
Accepted 5 November 2013
Available online 13 November 2013
Keywords:
2,3-Dihydroxybiphenyl 1,2-dioxygenase
Mesoporous silica
Immobilization
Molecular simulation
Biodegradation
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
effective catalytic cycle, BphC needs a chelated Fe (II) to transfer
electrons from the substrates to O2 to cleave the aromatic ring. But
Catechol and its derivatives are common intermediates dur-
ing the biodegradation process of aromatic compounds, which
can ultimately accumulate in the environment causing many seri-
ous environmental problems and harming human physical health
through dermal or respiratory track [1–4]. During the biodegra-
dation of catechol derivatives, the critical step is to cleave the
benzene ring by intradiol dioxygenases (ortho-cheavage) or extra-
diol dioxygenases (meta-cleavage) [5,6]. 2,3-Dihydroxybiphenyl
1,2-dioxygenase (BphC) is one of the typical extradiol dioxy-
genases, which can catalyze the incorporation of both oxygen
atoms from O2 into catecholics, resulting in the ring-open muconic
semialdehyde adducts [7,8]. Previous studies have proved the
important role of BphC involved in the degradation of polychlo-
rinated biphenyls (PCBs) by cleaving the benzene ring, as well as
the potential application of BphC in biosynthesizing high valuable
fine chemicals [9,10]. However, only a few studies have been per-
formed to develop the biodegradation or biosynthesis processes
using extradiol dioxygenases due to their instability [11]. For an
the catalytic center Fe (II) is sensitive to O2, which can be easily
oxidized to Fe (III) leading to the enzyme inactivation [12,13]. In
this regard, it is highly desirable to develop a method which could
both enhance the stability of extradiol dioxygenase and decrease
its sensitivity to O2 simultaneously.
Immobilization of an enzyme onto the suitable supports is an
effective approach to achieve biotechnological applications such as
separation, catalysis and sensors [2,14,15]. It is in favor of enhanc-
ing enzyme stabilization by strengthening its structural rigidity,
but the loading amount and residual activity of enzymes actually
depend on the type of immobilization matrix [16–18]. Up to now,
various supports have been reported to immobilize proteins, such
as sol–gels [19], hydrogels [20], organic microparticles [21], porous
and nonporous inorganic support [22]. For extradiol dioxygenases,
catechol 2,3-dioxygenase (C23O) have been successfully immobi-
lized in glyoxyl agarose beads [23] and calcium alginate hydrogels
[20] to improve the enzyme’s stability and thermostability effec-
tively. However, the former matrix was easy to distort the enzyme
conformation by the multi-point covalent cross-links, leading to
lower recoverable activity. The later matrix was difficult to con-
trol the pore size and distribution which limited the diffusion of
substrate into or out of the alginate beads.
∗
Corresponding author. Tel.: +86 411 84706251; fax: +86 411 84706252.
E-mail address: qyy@dlut.edu.cn (Y. Qu).
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.11.003

Y. Qu et al. / Journal of Molecular Catalysis B: Enzymatic 99 (2014) 136–142
137
Recently, much attention has been paid to the mesoporous
silicates (MPs) due to their ordered porous structures and nar-
row pore size distribution, which could be suitable to entrap
the biomolecules [24,25]. Moreover, MPs have large surface area,
thermal and mechanical stability, and controllable pore size [26].
Since the first report in 1996 [27], MPs have successfully immobi-
lized various commercially available enzymes such as lipase and
cytochrome c [28,29]. As a widely used MPs, SBA-15 is available to
encapsulate a wider range of proteins by virtue of its adjustable
pore size (5–30 nm), which is comparable with the size of pro-
tein [18]. Gómez et al. [30] have succeeded in immobilizing a high
molecular weight ˇ-glucosidase (130 kDa as dimer) onto SBA-15.
Zhou et al. [31] immobilized a C C bond hydrolase MfphA onto SBA-
15 using molecular simulation assisted strategy. Therefore, SBA-15
may also be a potential immobilization matrix for BphC.
In our previous studies, a bphC gene (915 bp) encoding 2,3-
dihydroxybiphenyl1,2-dioxygenase (BphC) from Dyella ginsengisoli
LA-4 was cloned and heterologously expressed in recombinant
Escherichia coli [32]. In this study, mesoporous silica SBA-15 was
utilized for the immobilization of BphC and molecular simulation
studies were performed to guide the immobilization process. The
catalytic performances of immobilized BphC, particularly the stor-
age and thermal stability, were investigated and compared with
that of the free enzyme. The possible mechanism for immobiliza-
tion of enzyme was proposed in the meantime. This study would
present more effective immobilization methods by mesoporous sil-
ica materials for metabolic enzymes, which could be widely used
during the process of biodegradation and bioremediation.
or immobilized enzyme was added. The molar extinction coeffi-
cient of HOPDA was determined as ε434 nm = 16,700 M−1 cm−1 at
pH 8.0. One unit of enzymatic activity was defined as the quantity
of enzyme required to form 1 ␮mol of HOPDA per minute.
2.4. Molecular simulation studies of BphC
Theoretical isoelectric point and free energy of folding of
BphC were calculated by Propka program based on the corre-
sponding protein sequence. According to the crystal structure
of a homologous protein, the three-dimensional model of BphC
was built and energy minimization was performed by YASARA
software, and the quality was evaluated by Structural Analy-
sis and Verification Server (http://nihserver.mbi.ucla.edu/SAVES/).
The active pocket was identified by the online CASTp server
(http://sts.bioengr.uic.edu/castp/calculation.php). APBS plugin for
Pymol were used to calculate the electrostatic surface of BphC at
pH value 8.0.
2.5. The catalytic performance of BphC immobilized on SBA-15
2.5.1. Kinetic parameters of free and immobilized enzymes
The Michaelis–Menten constant (Km) and catalytic turnover rate
(kcat) of the free and immobilized enzymes were calculated by
measuring the initial linear rates of the reaction after the addi-
tion of different concentrations of 2,3-dihydroxybiphenyl ranging
from 1 ␮M to 60 ␮M at ambient temperature. Three independent
measurements were carried out for each substrate concentration.
2.5.2. Effects of temperature and pH on the enzyme activity
2. Materials and methods
The free and immobilized enzymes were incubated in water
bath of different temperatures (from 15 ◦C to 70 ◦C) for 10 min
in Tris–HCl buffer (pH 8.0, 20 mM), then the residual activities
were determined. The effect of pH on the activity of immobilized
enzyme was investigated by incubating it in different buffers (pH
from 6 to 10) at 30 ◦C for 10 min. The activity of free enzyme
was investigated in the same condition as control. The pH of
buffer solutions used was as follows: 50 mM phosphate buffer
(pH 6.0–7.0), 50 mM Tris–HCl buffer (pH 8.0), 50 mM Tris–Glycine
buffer (pH 9.0–10.0). The extinction coefficients of HOPDA at pH
2.1. Chemicals
2,3-Dihydroxybiphenyl (>98% purity) was purchased from
Sigma-–Aldrich. Catechol and 3-methylcatechol were of 99% purity
and purchased from Tokyo Chemical Industry Co., Ltd. Other chem-
icals were of analytical grade. SBA-15 was synthesized using the
methodology described in previous work [28].
2.2. Enzyme extraction and immobilization
6–10 were 2000 M−1 cm−1, 11,300 M−1 cm−1, 16,700 M−1 cm−1
18,000 M−1 cm−1 and 17,400 M−1 cm−1, respectively.
,
E. coli BL21 (DE3), carrying plasmid pMD18-T containing the
gene bphC from D. ginsengisoli LA-4, was grown in Luria-Bertani (LB)
medium and the enzyme was purified by ÄKTA Explorer 100 (Amer-
sham Biosciences, Montreal, QC, Canada) as previously described
[32]. The concentration of purified BphC was determined by Brad-
ford method using bovine serum albumin as standard.
In order to perform immobilization, 3 ml purified enzyme solu-
tion was mixed with 2 mg SBA-15, which was dispersed uniformly
in 3 ml Tris–HCl (pH 8.0, 20 mM) by sonication for 10 min. The
final enzyme concentration was about 90 ␮g ml−1. After incubated
at 30 ◦C, 150 rpm for 5 h, samples were centrifuged and washed,
then suspended in Tris–HCl buffer to the same volume. The loading
amount of BphC was determined by recording the enzyme concen-
tration before and after immobilization experiments.
2.5.3. Storage stability and thermostability
Storage stability was investigated by monitoring the residual
activity of both free and immobilized enzymes every 12 h by stor-
ing them at 4 ◦C. The thermostability was investigated by assayed
their residual activity at regular interval at 40 ◦C, which was the
optimum temperature of free enzyme. Before assayed, the free and
immobilized enzymes were incubated 30 min at 40 ◦C water bath
for preheated.
2.5.4. Reusability
To determine the reusability of immobilized BphC, the residual
activity was assayed with 10 ␮M 2,3-dihydroxybiphenyl and 2 mg
immobilized BphC. After measuring the residual activity, the reac-
tion mixture was centrifuged and the supernatant was removed
between each cycle.
2.3. Activity assay
The activity of free and immobilized BphC were assayed by
monitoring the formation of cleavage product 2-hydroxy-6-oxo-
6-phenylhexa-2,4-dienoic acid (HOPDA) at 434 nm using a UV–vis
scanning spectrophotometer (V-560, JASCO, Tokyo, Japan). The
assay was performed in a reaction cuvette containing 10 ␮M 2,3-
dihydroxybiphenyl and 20 mM Tris–HCl buffer with a total volume
of 2.0 ml. The reaction was recorded immediately after the free
2.5.5. Catecholic compounds removal assay
Catecholic compounds (i.e., 2,3-dihydroxybiphenyl, catechol
and 3-methylcatechol) degradation studies were performed. Each
catechol and 3-methylcatechol (0.5 mM, 1.0 ml) was mixed with
1 mg of BphC-loaded SBA-15 in Tris–HCl buffer (9 ml, 50 mM, pH
8.0). Since 2,3-dihydroxybiphenyl could be instantly degraded by
BphC, the concentration of stock solution was increased to 1.0 mM,

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Y. Qu et al. / Journal of Molecular Catalysis B: Enzymatic 99 (2014) 136–142
and 1.0 ml stock solution was mixed with 0.05 mg BphC-loaded
SBA-15. They were all incubated at 30 ◦C, 100 r min−1 for 30 min.
The removal rates were determined by measuring the formation
of the ring-cleaving products at different time intervals at 434, 375
and 386 nm using UV–vis spectrophotometer. The molar extinction
coefficient for the products from catechol was 67,800 M−1 cm−1
from 3-methylcatechol was 73,600 M−1 cm−1 at pH 8.0.
,
2.6. Characterization of immobilized BphC
2.6.1. Fourier transformed infrared spectroscopy analysis
Fourier transformed infrared spectroscopy (FTIR) was used to
investigate whether the enzyme BphC had been immobilized on
SBA-15. The IR spectra of bare SBA-15 and enzyme-loaded SBA-
15 were obtained at ambient temperature using a Shimadzu IR
Prestige-21 FTIR spectrophotometer with the wavenumber ranging
from 400 to 3000 cm−1. Samples were prepared using the standard
KBr method with 0.3% and 3% sample in KBr (w/w).
Fig. 1. Theoretical free energy of folding (
) and titration curve (
) of
BphC at different pH. The protein was positive charge in the pH of the blue section
and the protein was negative charge in the pH of the red section.
2.6.2. N2 adsorption/desorption isotherms
N2 gas adsorption/desorption isotherms of SBA-15 and enzyme-
loaded SBA-15 were measured by QuadraSorb-SI instrument
(Quantachrome, Germany). Samples were pretreated by vacuum
dying at 55 ◦C to remove any H2O. The surface areas were mea-
sured using the Brunauer–Emmett–Teller (BET) method and the
pore size distribution was calculated by the thermodynamic-based
Barrett–Joyner–Halenda (BJH) on the adsorption and desorption
branches of the N2 isotherm.
Thus pH 8.0 could be suitable for obtaining higher activity of free
BphC, which was in consistent with the previous work [32]. On
the other hand, the predicted titration curve of BphC showed that
the theoretical isoelectric point was about 5.68. When pH was less
than 5.68, the protein was positive charged. According to the pre-
vious work, the isoelectric point for pristine SBA-15 was about 3.8.
Therefore, when pH was between 3.8 and 5.68, the SBA-15 was
negative charged and the BphC was positive charged, which was
benefit for the electrostatic interaction. When pH was larger than
5.68, the electrostatic interaction became weak with pH increasing.
Considering both loading amount and activity of free BphC, further
immobilization studies were performed between pH 6.0 and 10.0.
2.6.3. Determination of the metal Fe content by ICP-OES
The metal Fe content of free or immobilized BphC was measured
by inductively coupled plasma spectrometry optical emission spec-
troscopy (ICP-OES) metal analysis. Three samples were prepared:
purified enzyme solution, dry enzyme-loaded SBA-15 and con-
trolled SBA-15. The controlled SBA-15 was incubated in the same
culture condition but without enzyme to eliminate interference
of Fe element contained in the reaction system. The enzyme-
loaded SBA-15 and controlled SBA-15 were dried by vacuum drying
overnight. Then the dry samples were mixed with HF/HCl solu-
tion to dissolve over 8 h [33]. Meanwhile, the free enzyme solution
was digested in 5% HNO3 overnight and then removing precipitated
protein by centrifugation [34].
3.2. Immobilization of BphC on SBA-15
3.2.1. Fourier transformed infrared analysis
Fourier transformed infrared analysis (FTIR) was used to confirm
that BphC had been successfully immobilized on SBA-15. The FTIR
spectra of bare SBA-15 and enzyme-loaded SBA-15 were showed
in Fig. 2(A). Various vibrational modes of the silicate, Si OH and
Si
O
Si, were observed at 966 cm−1 and 802 cm−1, respectively.
As shown in the inset figure, the characteristic peak (amide II) of
protein loaded on SBA-15 was obviously observed in SBA-15-BphC
spectrum at 1534 cm−1, indicating a physical interaction between
the support and enzyme. Furthermore, the immobilized enzyme
and the purified enzyme were identified by sodium dodecyl sulfate
poly-acrylamide gel electrophoresis (SDS-PAGE) (Fig. 2(B)), which
also proved that the enzyme BphC was successfully immobilized
on SBA-15.
2.6.4. Circular dichroism (CD)
The circular dichroism spectra in the far-UV region
(200–260 nm) were analyzed to monitor the content of the regular
secondary structure of the free and immobilized BphC at room
temperature. The free protein concentration was 0.12 mg ml−1
in 5 mM Tris–HCl buffer at pH 8.0. The immobilized BphC was
acquired by mixing 0.18 mg ml−1 SBA-15 with the above free
protein solution for 5 min. The CD spectra were measured using
JASCO 810 (Tokyo, Japan) and each spectrum was performed in
three times. Spectrum of buffer blanks was measured prior to
sampling and digitally subtracted from CD spectra of samples.
3.2.2. N2 adsorption/desorption isotherms
The N2 adsorption/desorption measurements investigated the
textural features of the fiber-like SBA-15 before and after adsorp-
tion of BphC. The obtained isotherms can be classified as type IV
and exhibit an H1-type hysteresis loop at high relative pressure,
which illustrated the mesoporous material SBA-15 had typical fea-
tures of cylindrical channel. This result was the same as reported
by Salis et al. [35] and Zhou et al. [31]. Compared with the pris-
tine SBA-15, the BphC-loaded SBA-15 showed a marked decrease in
both the BET surface area (SBET) from 294.6 m2 g−1 to 168.4 m2 g−1
and pore volume from 0.34 cc g−1 to 0.24 cc g−1. The BJH pore size
of SBA-15 before and after immobilized BphC was about 3.405 nm
and 3.433 nm, respectively. The apparent decreases in the SBET and
pore volume suggested that some enzyme molecules might enter
3. Results and discussion
3.1. Molecular simulation studies of BphC
Activity and loading amount of BphC were both important
indexes for choosing the suitable immobilization condition. Before
immobilization, folding free energy and titration curve of BphC
were predicted through its primary structure. The free energy of
folding indicated the stability of protein. According to Fig. 1, BphC
could be stable between pH 5 and 10, and had the highest stabil-
ity at pH 8.0 (corresponding to the lowest free energy of folding).

Y. Qu et al. / Journal of Molecular Catalysis B: Enzymatic 99 (2014) 136–142
139
Fig. 2. (A) FTIR spectra of bare SBA-15 (black line) and enzyme-loaded SBA-15 (red
line) with 0.3% SBA-15 in KBr (w/w). The inset showed the enlarge region around
amide II peak, with 3% SBA-15 in KBr (w/w). (B) SDS-PAGE results of immobilized
BphC: lane E crude extracts; lane F free BphC; lane I immobilized BphC; lane M protein
molecular weight marker. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
Fig. 3. The effects of different temperature (A) and pH (B) on the activity of free
(
) and immobilized (
) BphC.
into the tunnels of SBA-15. The similar results have been reported
for the immobilization of lipase onto SBA-15 [33,34].
enzymatic activity of BphC loaded on SBA-15 was about 38%, which
was similar to other immobilized dioxygenases [20].
3.2.3. The effect of pH on the immobilization process
The purified BphC was incubated with 2 mg SBA-15 in buffer
solutions with different pH values ranging from 6.0 to 10.0 for
3.5 h. It was shown that the maximum loading amount of BphC
was 152.26 7.62 mg g−1 at pH 6.0 (Table 1). And the loading
amount of BphC decreased along with the increase of pH values,
which could be explained by the molecular simulation mentioned
above. During the pH ranging from 6 to 10, both enzyme molecule
and mesoporous silica were negative-charged. When pH was ris-
ing, the zeta-potential of SBA-15 increased rapidly, resulting in an
increase of the electrostatic repulsion between enzyme and SBA-
15. In the case of specific activity, when BphC was immobilized
at pH 8.0, immobilized enzyme had the highest specific activity of
10.74 0.64 U mg−1, and it also had an acceptable loading amount
of 103.20 8.12 mg g−1. Therefore, pH 8.0 was the optimal condi-
tion considering the equilibrium of loading amount and activity of
enzyme.
3.3. The catalytic performance of BphC immobilized on SBA-15
3.3.1. Determination of the kinetic parameters of
2,3-dihydroxybiphenyl
The activities of free and immobilized BphC were measured
at different concentrations of 2,3-dihydroxybiphenyl in order to
calculate the values of Km and kcat. The values of Km and kcat
for free enzyme were 3.64 ␮M and 79.56 s−1, while for immobi-
lized enzyme were 10.22 ␮M and 20.63 s−1, respectively. About
3-folds increase in Km value from 3.64 to 10.22 ␮M was found
for immobilized enzyme, whereas the kcat value was decreased on
immobilization of BphC compared to the free form. The apparent
increase of Km and decrease of kcat might be due to the conformation
change of enzyme molecules and diffusion limitation of substrate
into the pore of SBA-15.
The immobilization process was further optimized. The adsorp-
tion equilibrium was reached after incubating at pH 8.0 for 5 h. The
final loading amount was 124.6 mg protein g−1 support, which was
higher than that of the previous report [37]. Nardo et al. immo-
bilized catechol 1,2-dioxygenase from Acinetobacter radioresistens
S13 on nanosponges and obtained a maximum loading amount
of only 28.9 mg protein g−1 support. Meantime, the remained
3.3.2. Effects of temperature and pH on the enzyme activity
The effect of temperature on free and immobilized enzyme
activity was determined over a wide range of 15–70 ◦C. As shown
in Fig. 3(A), the immobilized enzyme exhibited its suitable tem-
perature region from 15 to 55 ◦C with the highest activity at 45 ◦C,
while the free enzyme showed the highest activity at 40 ◦C and

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Y. Qu et al. / Journal of Molecular Catalysis B: Enzymatic 99 (2014) 136–142
Table 1
The effects of different pH values on the immobilization process within 3.5 h.
pH values
6.0
7.0
8.0
9.0
10.0
Loading amount (mg g−1
Specific activity (U mg−1
)
)
152.26 7.62
2.56 0.23
125.12 5.27
7.75 0.39
103.20 8.12
10.74 0.64
61.72 4.20
8.40 0.54
41.02 2.15
0.59 0.17
Fig. 5. Long term usability of immobilized BphC during nine cycles.
the free enzyme remained only 0.8% of its initial activity after 12 h,
whereas the activity of the immobilized enzyme could maintain
16% of the initial activity after 5 days.
The thermostability of the free and immobilized enzyme was
measured by incubating at 40 ◦C (the optimal temperature for free
enzyme) over time. Fig. 4(B) shows that the free enzyme retained
67.8% of the initial activity after 3 h, while the immobilized form
still maintained its initial activity. In the case of the free enzyme,
almost all of the initial activity was lost after 15.5 h, whereas the
immobilized enzyme retained 24.7% of the maximal activity. Our
results were similar with the previous reports about the intradiol
and extradiol dioxygenase entrapped or covalently immobilized
on other supports [20,23,37]. It was revealed that the interaction
between enzyme and support could increase conformational sta-
bility.
3.3.4. Reusability
Fig. 4. The stability of immobilized BphC. (A) The storage stability of free (
One of the primary advantages of immobilization enzymes in an
industrial process is the reusability. The reusability of immobilized
BphC was monitored at room temperature, in which it was obvious
that there existed an activity decrease for each cycle (Fig. 5). After
nine continuous cycles, the immobilized enzyme retained 9.0% of
initial activity. One of the factors was the loss of the loaded enzyme
during separating the supernatant and sediment, the other rea-
son was the weak electrostatic interaction between enzyme and
support SBA-15 by direct physical adsorption.
) and immobilized (
immobilized (
) BphC at 4 ◦C; (B) Thermostability of free (
) and
) BphC at 40 ◦C.
only retained 42.5% of its activity at 55 ◦C. The effects of pH val-
ues on the relative activity of the free and immobilized BphC were
shown in Fig. 3(B) within the pH range of 6.0–10.0. It was clearly
revealed that both the free and immobilized enzymes exhibited the
maximum activity at pH 8.0.
3.3.3. Storage stability and thermostability
3.3.5. Catecholic compounds removal assay
Storage stability is one of the most important parameters to be
considered during the process of enzyme immobilization. The free
enzymes dramatically lost their activities because of the inhibitor
oxygen molecule. The stability of the immobilized BphC was deter-
mined by storing the suspension of immobilized enzyme in pH 8.0
Tris–HCl buffer (20 mM) at 4 ◦C. Compared with the free enzymes,
the activity of immobilized enzyme could last much longer under
the same storage conditions (showed in Fig. 4(A)). The activity of
In order to assess the applicability of immobilized BphC on the
wastewater treatment, the efficiency of the catecholic compounds
removal was tested by adding the immobilized BphC. As shown
in Fig. 6, the removal percentages of 2,3-dihydroxybiphenyl, cate-
chol and 3-methylcatechol were about 71.73%, 24.55% and 30.11%,
respectively. The removal rates of 2,3-dihydroxybiphenyl and cat-
echol were obviously decreased after 10 min, while the removal
rate of 3-methylcatechol was instantly decreased in 5 min. This

Y. Qu et al. / Journal of Molecular Catalysis B: Enzymatic 99 (2014) 136–142
141
revealed that the decrease of activity was not due to the change of
Fe content of the immobilized BphC.
3.4.2. Circular dichroism experiment
The circular dichroism (CD) is very sensitive to examine the sec-
ondary structure of proteins [29]. Therefore, the CD spectrum of
BphC onto mesoporous silica SBA-15 was compared with that of the
free enzyme in the far-UV region, revealing the effect of weak physi-
cal interaction for the protein structure. Fig. 7 shows that the far-UV
CD spectrum of the immobilized BphC was quite different from
that of the free enzyme. The immobilized enzyme had a detectable
change, which the ˛-helix content was noticeably reduced and
the ˇ-sheet content was appeared. It was demonstrated that the
secondary structure of BphC was significantly altered after being
immobilized on SBA-15 with physical adsorption, which most likely
enhanced the stabilization of enzyme, but also caused the partial
deactivation of enzyme molecules [35,39].
4. Conclusion
The molecular simulation approach was introduced to ana-
lyze the immobilization process of BphC onto SBA-15. And the
enzyme was successfully immobilized onto SBA-15 through the
identification of FTIR and N2 adsorption/desorption isotherms.
By immobilization, the enzyme storage stability and thermosta-
bility over a wide range of temperatures were both improved.
Meanwhile, the immobilized enzyme could be reused nine times
with 9.0% residual activity. Decrease of relative activity was
due to the secondary structure change during the immobiliza-
tion. This study provides some fundamental information about
2,3-dihydroxybiphenyl 1,2-dioxygenase immobilized on SBA-15,
which should expand the application range of this enzyme in both
biodegradation and biosynthesis process.
Fig. 6. The efficiency of immobilized BphC for removal of 2,3-dihydroxybiphenyl
), catechol ( ) and 3-methylcatechol ( ) in the batch system.
(
performance was probably due to the inhibition of products accu-
mulated in the reaction process, which was reported by the
previous studies [20].
3.4. Proposed mechanism of immobilization
3.4.1. Metal analysis of BphC
Compared with the native enzyme, the immobilized BphC on
SBA-15 had a relatively low retention activity (38%). The chelated
Fe ion in the activity site was essential for the effective enzymatic
catalysis. Therefore, the chelated Fe content before and after immo-
bilization was detected by ICP-OES in order to determine the effects
of the nanomaterial for activity on the immobilization process. The
results suggested that the purified BphC contained 0.91 iron atoms
per enzyme molecule, equivalent to a protein/Fe ratio of 1:1. Mean-
while, the iron content of loaded BphC was found to be approximate
at 1.02 iron atoms per enzyme molecule, suggesting that iron con-
tent of BphC after immobilization did not significantly lost. It was
Acknowledgement
This work was supported by the National Natural Science Foun-
dation of China (51078054, 21176040, and 20923006).
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.molcatb.
2013.11.003.
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